CA1081209A - Process for the preparation of peptides - Google Patents
Process for the preparation of peptidesInfo
- Publication number
- CA1081209A CA1081209A CA235,873A CA235873A CA1081209A CA 1081209 A CA1081209 A CA 1081209A CA 235873 A CA235873 A CA 235873A CA 1081209 A CA1081209 A CA 1081209A
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- CA
- Canada
- Prior art keywords
- acid
- formula
- alpha
- group
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Cephalosporin Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
An improved process for producing a peptide which comprises treating a phosphoramide derivative of the formula:
An improved process for producing a peptide which comprises treating a phosphoramide derivative of the formula:
Description
~ V~3 B~CKGROUND OF T~IE INVENTION
_______ 1. Field of the Invention The present invention relates to an improved process for producing a peptide. ~lore par~icularlv, it relates to an ~ ~;
improved process ~or elimination o~ a p~otective phosnhoryl group in producing.a.peptide.
_______ 1. Field of the Invention The present invention relates to an improved process for producing a peptide. ~lore par~icularlv, it relates to an ~ ~;
improved process ~or elimination o~ a p~otective phosnhoryl group in producing.a.peptide.
2. Description of the Prior Art In general, in synthetic reactions of a compound con- :
taining a functional group, it is necessary to protect the functional group from taking part in the reaction depending on the object of the reaction, and then to remove the protective :
group selectively after the desired reaction is completed. : ~
Particularly, in the synthesis of the peptide of the formula (I) ~ -described herein, it is essent.ial to protect th~e amino group. .
and to remove the protective group. .~ ~:
. ; . .
An example in which the phosphoryl group of the formula ` ~
O : .
RlOP- was used as a pro-tective group for an amino.group in the ~:
O-R ::
synthesis of a peptide, is disclosed in L. Zervas et al., Chem. ~.
Ber. 94, 2644 (1961). L. Zervas et al. used dibenzylphosphoryl ~ ~
or para-substituted dibenzylphosphoryl groups as a prote~tive .``
group for an amino group of an amino acid, and for the purpose of dephosphorylation emplo~ed the following two methods:
(A) treatment of.the N-phosphoryl peptide w.ith hydrogen bromide -.
in a solvent, and (B) catalytic hydrogenation.
The method of treatment of the N-phosphor~l peptide :-with hydrogen bromide in a solvent, however, includes many problems, because of the high acidity of hydrogen bromide, in 8~9 1 the preparation of a peptide wh:ich is unstable to acids. For example, a study recently made showed that, when this method was applied to the pr~paration of a peptide including a cephalo-sporin, it was difficult to avoid various side-reactions such as cleavage of the ~~lactam riny in the cephalosporin nucleus.
Therefore, this method is not suitable for this purpose.
On the other hand, the use of an expensive palladium catalyst is essential for the catalytic hydrogenation method, and moreover in the case of a peptide which contains a sulur-containing amino acid such as cysteine and methionine as disclosed in, Helv. Chim. Acta., 42, 1257(1959), the catalyst is often easily poisoned and the catalytic hydrogenation ceases.
Furthermore, as described in Chem. Ber., 94, 2644 (1961) cited above, phosphoryl groups other than a dibenzyl-phosphoryl group or its para-substituted derivatives, for example, dialkylphosphoryl groups such as a diethylphosphoryl group, are not dephosphorylated by either of the methods (A) and (B) above.
Dephosphorylation of N-phosphoryl groups contained in the above-mentioned peptides including a cephalosporin is disclosed in ~ Dutch Patent No. 7,200,432, and specifically 7-[5'-carboxy-5'-(diphenylphosphoramido)valeramidol-3-acetoxymethyl-ceph-3-em-4- ~ -carboxylic acid is reacted with sodium acetate in methanol to obtain sodium 7-[5'-carboxy-5'-aminovaleramido~-3-acetoxymethyl-ceph-3-em-4-carboxylate. Butl as the result of a study recently made, it has been found that this dephosphorylation method can not be employed for the dephosphorylation of all compounds of the formula (II) described herein.
SUMMARY OF THE INVENTION
_ _ _ _ _ _ _ _ One object of the present invention is to provide an advantageous process for preparing various peptides.
.
1 Further objects will be apparent Erom -the following description.
These objects are achieved by a process for the elimination of a protective phosphoryl group of the formula O
RlO - P - (III) wherein Rl and R2 are each an al]cyl group, a cycloalkyl group~
an aryl group or an aralkyl group, from a phosphoramide . ; -derivatlve of the formula (II): . :
o RlO - I - NH - Al (II) .
2 ~.
~ .
wherein Rl and R2 are as defined above and Al is a peptide residue to produce a peptide of the formula (I):
' Al ~ NH2 tI) wherein Al is as defined above by treating the phosphoramide derivative of the formula (II) with a phosphorus acid.
Thus, the invention provides a process for producing a peptide of the formula (I): :-Al - NH2 (I) :~:
,
taining a functional group, it is necessary to protect the functional group from taking part in the reaction depending on the object of the reaction, and then to remove the protective :
group selectively after the desired reaction is completed. : ~
Particularly, in the synthesis of the peptide of the formula (I) ~ -described herein, it is essent.ial to protect th~e amino group. .
and to remove the protective group. .~ ~:
. ; . .
An example in which the phosphoryl group of the formula ` ~
O : .
RlOP- was used as a pro-tective group for an amino.group in the ~:
O-R ::
synthesis of a peptide, is disclosed in L. Zervas et al., Chem. ~.
Ber. 94, 2644 (1961). L. Zervas et al. used dibenzylphosphoryl ~ ~
or para-substituted dibenzylphosphoryl groups as a prote~tive .``
group for an amino group of an amino acid, and for the purpose of dephosphorylation emplo~ed the following two methods:
(A) treatment of.the N-phosphoryl peptide w.ith hydrogen bromide -.
in a solvent, and (B) catalytic hydrogenation.
The method of treatment of the N-phosphor~l peptide :-with hydrogen bromide in a solvent, however, includes many problems, because of the high acidity of hydrogen bromide, in 8~9 1 the preparation of a peptide wh:ich is unstable to acids. For example, a study recently made showed that, when this method was applied to the pr~paration of a peptide including a cephalo-sporin, it was difficult to avoid various side-reactions such as cleavage of the ~~lactam riny in the cephalosporin nucleus.
Therefore, this method is not suitable for this purpose.
On the other hand, the use of an expensive palladium catalyst is essential for the catalytic hydrogenation method, and moreover in the case of a peptide which contains a sulur-containing amino acid such as cysteine and methionine as disclosed in, Helv. Chim. Acta., 42, 1257(1959), the catalyst is often easily poisoned and the catalytic hydrogenation ceases.
Furthermore, as described in Chem. Ber., 94, 2644 (1961) cited above, phosphoryl groups other than a dibenzyl-phosphoryl group or its para-substituted derivatives, for example, dialkylphosphoryl groups such as a diethylphosphoryl group, are not dephosphorylated by either of the methods (A) and (B) above.
Dephosphorylation of N-phosphoryl groups contained in the above-mentioned peptides including a cephalosporin is disclosed in ~ Dutch Patent No. 7,200,432, and specifically 7-[5'-carboxy-5'-(diphenylphosphoramido)valeramidol-3-acetoxymethyl-ceph-3-em-4- ~ -carboxylic acid is reacted with sodium acetate in methanol to obtain sodium 7-[5'-carboxy-5'-aminovaleramido~-3-acetoxymethyl-ceph-3-em-4-carboxylate. Butl as the result of a study recently made, it has been found that this dephosphorylation method can not be employed for the dephosphorylation of all compounds of the formula (II) described herein.
SUMMARY OF THE INVENTION
_ _ _ _ _ _ _ _ One object of the present invention is to provide an advantageous process for preparing various peptides.
.
1 Further objects will be apparent Erom -the following description.
These objects are achieved by a process for the elimination of a protective phosphoryl group of the formula O
RlO - P - (III) wherein Rl and R2 are each an al]cyl group, a cycloalkyl group~
an aryl group or an aralkyl group, from a phosphoramide . ; -derivatlve of the formula (II): . :
o RlO - I - NH - Al (II) .
2 ~.
~ .
wherein Rl and R2 are as defined above and Al is a peptide residue to produce a peptide of the formula (I):
' Al ~ NH2 tI) wherein Al is as defined above by treating the phosphoramide derivative of the formula (II) with a phosphorus acid.
Thus, the invention provides a process for producing a peptide of the formula (I): :-Al - NH2 (I) :~:
,
- 3 ~
`:
- 10~ 9 1 wherein ~1 is ~ peptide residue derived from a peptide, comprising treating a phosphoramide derivative of the ~ormula (II):
R10 - I - NH - Al (II) .
wherein Rl and R2 are each an alkyl group, a cycloalkyl group, an aryl group or an aralkyl group, with a phosphorus acid. :
DETAILED DESCRIPTION OF THE INVENTION
In order to overcome the defects in conventional processes, various studies were conducted and it was found that a group represented by the ~ormula (III):
O
Il ' ' - ~ .
wherein Rl and R2 are as defined hereinbefore, is stable during the course of synthesis of peptides, and can be readily removed with a phosphorus acid under mild conditions, and there~ore that the group can advantageously be used as a protective group during the synthesis of a peptide.
That is, as the dephosphorylation according to the pxesent invention is carried out under a very mild condition, ::
even an unstable peptide, for example; the above-described ~ :
ones including cephalosporins, can be dephosphorylated without side-reactions such as cleavage of the ~-lactam ring of the cephalosporin nucleus occurring. Furthermore, in the present
`:
- 10~ 9 1 wherein ~1 is ~ peptide residue derived from a peptide, comprising treating a phosphoramide derivative of the ~ormula (II):
R10 - I - NH - Al (II) .
wherein Rl and R2 are each an alkyl group, a cycloalkyl group, an aryl group or an aralkyl group, with a phosphorus acid. :
DETAILED DESCRIPTION OF THE INVENTION
In order to overcome the defects in conventional processes, various studies were conducted and it was found that a group represented by the ~ormula (III):
O
Il ' ' - ~ .
wherein Rl and R2 are as defined hereinbefore, is stable during the course of synthesis of peptides, and can be readily removed with a phosphorus acid under mild conditions, and there~ore that the group can advantageously be used as a protective group during the synthesis of a peptide.
That is, as the dephosphorylation according to the pxesent invention is carried out under a very mild condition, ::
even an unstable peptide, for example; the above-described ~ :
ones including cephalosporins, can be dephosphorylated without side-reactions such as cleavage of the ~-lactam ring of the cephalosporin nucleus occurring. Furthermore, in the present
- 4 -'~ ;;
:. , , ~ .
1 inven tiOIl, the above~clescribed phosphoryl group can be used in either case where the substituen-ts, Rl and R2, are an alkyl group, a cycloalkyl group, an aryl group or an aralkyl yroup, and moreover can be used industrially advantageously because of the use of an inexpens~ve phosphorus acid.
In the above general formulae, Rl and R2 are each a Cl - C6 alkyl group such as methyl, ethyl, isopropyl, ~ -n-butyl or n-amyl; a C3 - C7 cycloalkyl group such as tO cyclohexyl or cyclopentyl; an aryl group such as phenyl, p-tolyl or p-chlorophenyl; and an aralkyl group such as benzyl, p-nitrobenzyl or p-chlorobenzyl; and the like.
The peptide represented by the formula (I) designates a compound which includes at least two amino `
acids connected to each other through a peptide linkage.
The peptide residue represented by the formula -Al in the formula (I) means a group formed by eliminating an amino group from the peptide of the formula (I). -The amino acids which make up the peptide can be any natural, synthetic and fermentation products, and further can be in any of an L-, D- and DL- forms. Speci-fic examples of such amino acids include, ~or instance, glycine, alanine, valine, norvaline, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, sarcosine, aspartic aci~, asparagine, glutamic acid, glutamine, lysine, ornithine, arginine, phenylalanine, tyrosine, histidine, triptophan, proline, hydroxyproline, a-aminobutyric acid, ~-aminobutyric acid, ~,y-diaminobutyric acid, ~-aminoadipic acid, ~-phenyl-glycine, ~-p-hydroxyphenylglycine, a-p-chlorophenylglycine, a-(2-thienyl)-glycine, ~-(2-furyl)glycine, ~-(l-cyclohexenyl) ~ . . . _ ~
:~08~
1 glycine, and N~ cyclohexadienyl)c31ycine.
The t~rm "amino acid" as used herein also includes 7-amino-~-carboxy-3-methyl-3-cephem. [l~ith respect to the nomenclature for the 3-cephem compound, reference is made to the Journal of the ~merican Chemical Society, 84, 340 (1962).J
The peptide representecl by the formula (I) also includes a peptide in which other functional groups, if any, (e.g., amino, hydroxylr carboxyl, mercapto, etc.) are protected with other conventional protec-tive groups commonly employed in the art of the present invention.
The peptide represented by the formula (I), `
~1 ~ NH2 are well-known substances useful as a medicine or ~ ~ `
a precursor thereof, and particularly, the cephalosporin is well-known as a potent antimicrobial agent or a pre-cursor thereof.
The term "phosphorus acid" is employed herein to describe phosphorus acid type compounds such as ortho-phosphoric acid, phosphorous acid, phosphonic acid, phosphinic acid, and the ester and anhydride derivatlves thereof. Specifically t suitable derivatives include phosphoric es~ers such as monomethyl phosphate, dimethyl phosphate, monoethyl phosphate, monophenyl phosphate, diphenyl phosphate and monobenzyl phosphate; phosphorous esters such as monomethyl phosphite and monophenyl phosphite; phosphoric acid anhydrides such as pyrophosphoric acid, polyphosphoric acid ~nd phosphorus pentoxide; phosphoric ester anhydrides such as dimethyl pyrophosphate, diphenyl pyrophosphate and polyphosphoric ester; phosphonic acid derivatives such as methylphosphonic acid and phenylphosphonic acid; and phosphinic acid derivatives such 3~ as dimethylphosphinic acid and diphenylphosphinic acid. Of these .,~.., . !
: ` ' . . ~ ,, , ~ O 9 1 phosphorus ~cids, ortho-phosphoric acid, phosphorous acid, and polyphosphoric acid are par-ticularly preferred indu~trially.
In carrying out the process of the presenk invention, the amount of phosphorus acid used in the reaction can vary from the phosphorus acid beiny present in excess to the compound of the formula (II) being present in excass, bu-t an ~mount of more than 1 mole per mole of the compound of the formula ~II), particularly, more than 3 moles is particularly preferred for obtaining good results. And since the phosphorus acid can act as the reaction medium, the amount o~ the phosphorus acid can range up to about 100 to S00 moles per mole of the compound of the formula (II).
According to the invention, the elimination of the phosphoryl group of the formula (III) is carried out by reacting the phosphoramide derivative of the formula (II) with a phosphorus acid in the absence of a solvent, or in a solution or suspension in an inert solvent.
When an inert solvent is employed, the amount of the inert solvent is such that the concentration of the phosphorus acid is more than about 10% by weight, but a concentration of 50 to 100~ by weight (i.e., no solvent) is preferred particularly for obtaining good results.
Preferred examples of the inert solvents are as follows:
aromatic hydrocarbons, e.g., benzene, toluene, etc.; chlorinated hydrocarbons, e.g., dichloromethane, chloroform, etc.; ethers, e.g., dioxane, diethyl ether, etc.; alcohols, e.g., methanoli amides such as dimethylformamide, etc.; dimethylsulfoxide; water;
or carboxylic acids such as formic acid, acetic acid, propionic acid, etc.
,~
. . ~ . - .
, 1 The reaction suitably proceeds at temperature above about -20C, bu-t in general, a temperature ranye between 0C and 70C is preferred for obtaining good results. Generally, the reaction is completed in a period of about 2 to 50 hours.
Applying the process of the present invention, the peptide of the formula (I) can be prepared in high yield by pr~tecting the amino group in an amino acid, peptide or derivative thereof temporarily with the phosphoryl group of the formula (III), con-verting them to the desired peptides which still have this pro-tective group, and then eliminating the amino protective group with a phosphorus acid under mild conditions.
Furthermoxe, as is weIl known in general, it is very important for ~solation and purification of a compound containing functional group to protect the functional group temporarily, and in this respect the present invention is also very useful for achieving such a purpose. For example, in order to obtain a compound of the formula (I) in high purity, this purification can be achieved by converting the impure compound of the formula (I) to the phosphoryl derivative of the formula (II) by a well-known method, purifying the phosphoryl derivative of the formula (II), and then de-phosphorylating the derivative of the formula (II! to the compound of the formula (I) by the process of this invention.
The peptide of the formula (II) can be prepared by a conventional process per se in the art of the present invention, ;~
for instance, they can be prepared by reacting an amino acid or ~`~
a peptide of the formula (IV)~
A2 ~ NH2 (IV) 1083~)9 1 which consti-tutes the peptide of the formula (I) together with an additional amino acid or peptide or which is the peptide of the formula (I), with a compound of the formul~ (v):
R10 ~ P ~ X . (V) ~ , wherein Rl and R2 are each as defined above; and X is a halogen atom, to produce a compound of the formula (VI):
O
R 0 - P - NH - A ~VI) OR2 ~: , wherein Rl and R2 are each as defined above; and A2 is the residual group of the amino acid or peptide o~ the formula (IV), and, if necessary, then expanding ~2 to Al by condensation of the compound oE the formula (VI) with an additional amino acid or peptide.
The condensation can be carried out employing various condensation methods which are used in the axt, for example, an acid halide method, a mixed acld anhydride method and a reactive ester method.
In the synthesis of the compounds represented by formulas (II) and (IV), it is of course necessary to previously protect functional groups which adversely affect the desired reaction with a suitable protective group as used in the art.
The following examples are given to illustrate the invention more specifically, but are not in any way to be construed as limiting the scope o~ the present invention.
3~ Unless otherwise indicated herein, all parts, percents, ratios and the like are by weight. ~-~ _ 9 _ ~;)81~09 1 FX~MPLE`~ l Preparation of 7-(D-a-Aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylic ~cid Step A:
In llO mQ of an aqueous lN-sodium hydroxide solution was dissolved 15 g of D-phenylglycine. To the resulting solution was added dropwise 20 g of diJnethylphosphoryl chloride at room temperature (i.e., 20~ 30C) while keeping the pH of the reaction mixture at 9 to ll with the addition o an aqueous 2N-sodium hydroxide solution.
The reaction mixture was stirred for an additional 20 minutes, washed with diethyl ether, adiusted to a pH of 2 with conc. hydrochloric acid and then extracted with dichloromethane.
The organic layer was washed with water, dried over magnesium sulfate and concentrated to obtain 16 g of oily D-a-(dimethyl-phosphoramido)phenylacetic acid.
Q ) IR: vmax (neat) 1725 cm 1 Three grams of the product thus-obtained was dissolved ~ .
in 30 mQ of dichloromethane and then 3 g of thionyl chloride was ~ ~;
added thereto. The resulting solution was heated under reflux for 1 hour. The reaction solution was concentrated under reduced pressure to obtain 3.2 g of oily D-~-(dimethylphosphoramido)phenyl~
acetyl chlorlde. The product thus-obtained showed a strong absorption due to a carbonyl group (-COCQ) at 1805 cm l in the IR spectrum.
To a suspension of 2 g of 7~amino-3-methyl-ceph-3-em-4-carboxylic acid in 30 mQ of water 2.4 g of sodium bicarbonate was added to dissolve the acid. The resulting solution was cooled to 0 to 5C and a solution of 3 g of the acid chloride 1 ~ -.
.... . . . . , . . . . ,,, .. , ,, ~ . .... .. .
10~1~(19 1 obtained above in 20 rnQ of ace-tone was added dropwise thereto while stirrincJ. The reaction mixture was stirred for an additional 20 minutes, adjusted ~o a pll of 1 with addition of conc. hydro-chloric acid and extracted with dichloromethane. The separated dichloromethane layer was dried over magnesium sulfate and concentrated. Diethyl ether was added to the residue and the precipitated crystals were filtered to obtain 2.3 g of 7-[D-a-(dimethylphosphoramido)phenylacetamido]-3-methyl-ceph-3-em-4-carboxylic acid (m.p. 190 ~ 192C).
1CI :
[~D~ +78 (c = 1, EtOH) IR: vmax (Nujol) 1780, 1715, 1660 cm Step B:
Two grams of 7-[D-~-(dimethylphosphoramido)phenyl-acetamido]-3-methyl-ceph-3-em-4-carboxylic acid which wa~ obtained in Step A above was dissolved in 5 mQ o~ 85% phosphoric acid, and the resulting mixture was stirred at room temperature for
:. , , ~ .
1 inven tiOIl, the above~clescribed phosphoryl group can be used in either case where the substituen-ts, Rl and R2, are an alkyl group, a cycloalkyl group, an aryl group or an aralkyl yroup, and moreover can be used industrially advantageously because of the use of an inexpens~ve phosphorus acid.
In the above general formulae, Rl and R2 are each a Cl - C6 alkyl group such as methyl, ethyl, isopropyl, ~ -n-butyl or n-amyl; a C3 - C7 cycloalkyl group such as tO cyclohexyl or cyclopentyl; an aryl group such as phenyl, p-tolyl or p-chlorophenyl; and an aralkyl group such as benzyl, p-nitrobenzyl or p-chlorobenzyl; and the like.
The peptide represented by the formula (I) designates a compound which includes at least two amino `
acids connected to each other through a peptide linkage.
The peptide residue represented by the formula -Al in the formula (I) means a group formed by eliminating an amino group from the peptide of the formula (I). -The amino acids which make up the peptide can be any natural, synthetic and fermentation products, and further can be in any of an L-, D- and DL- forms. Speci-fic examples of such amino acids include, ~or instance, glycine, alanine, valine, norvaline, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, sarcosine, aspartic aci~, asparagine, glutamic acid, glutamine, lysine, ornithine, arginine, phenylalanine, tyrosine, histidine, triptophan, proline, hydroxyproline, a-aminobutyric acid, ~-aminobutyric acid, ~,y-diaminobutyric acid, ~-aminoadipic acid, ~-phenyl-glycine, ~-p-hydroxyphenylglycine, a-p-chlorophenylglycine, a-(2-thienyl)-glycine, ~-(2-furyl)glycine, ~-(l-cyclohexenyl) ~ . . . _ ~
:~08~
1 glycine, and N~ cyclohexadienyl)c31ycine.
The t~rm "amino acid" as used herein also includes 7-amino-~-carboxy-3-methyl-3-cephem. [l~ith respect to the nomenclature for the 3-cephem compound, reference is made to the Journal of the ~merican Chemical Society, 84, 340 (1962).J
The peptide representecl by the formula (I) also includes a peptide in which other functional groups, if any, (e.g., amino, hydroxylr carboxyl, mercapto, etc.) are protected with other conventional protec-tive groups commonly employed in the art of the present invention.
The peptide represented by the formula (I), `
~1 ~ NH2 are well-known substances useful as a medicine or ~ ~ `
a precursor thereof, and particularly, the cephalosporin is well-known as a potent antimicrobial agent or a pre-cursor thereof.
The term "phosphorus acid" is employed herein to describe phosphorus acid type compounds such as ortho-phosphoric acid, phosphorous acid, phosphonic acid, phosphinic acid, and the ester and anhydride derivatlves thereof. Specifically t suitable derivatives include phosphoric es~ers such as monomethyl phosphate, dimethyl phosphate, monoethyl phosphate, monophenyl phosphate, diphenyl phosphate and monobenzyl phosphate; phosphorous esters such as monomethyl phosphite and monophenyl phosphite; phosphoric acid anhydrides such as pyrophosphoric acid, polyphosphoric acid ~nd phosphorus pentoxide; phosphoric ester anhydrides such as dimethyl pyrophosphate, diphenyl pyrophosphate and polyphosphoric ester; phosphonic acid derivatives such as methylphosphonic acid and phenylphosphonic acid; and phosphinic acid derivatives such 3~ as dimethylphosphinic acid and diphenylphosphinic acid. Of these .,~.., . !
: ` ' . . ~ ,, , ~ O 9 1 phosphorus ~cids, ortho-phosphoric acid, phosphorous acid, and polyphosphoric acid are par-ticularly preferred indu~trially.
In carrying out the process of the presenk invention, the amount of phosphorus acid used in the reaction can vary from the phosphorus acid beiny present in excess to the compound of the formula (II) being present in excass, bu-t an ~mount of more than 1 mole per mole of the compound of the formula ~II), particularly, more than 3 moles is particularly preferred for obtaining good results. And since the phosphorus acid can act as the reaction medium, the amount o~ the phosphorus acid can range up to about 100 to S00 moles per mole of the compound of the formula (II).
According to the invention, the elimination of the phosphoryl group of the formula (III) is carried out by reacting the phosphoramide derivative of the formula (II) with a phosphorus acid in the absence of a solvent, or in a solution or suspension in an inert solvent.
When an inert solvent is employed, the amount of the inert solvent is such that the concentration of the phosphorus acid is more than about 10% by weight, but a concentration of 50 to 100~ by weight (i.e., no solvent) is preferred particularly for obtaining good results.
Preferred examples of the inert solvents are as follows:
aromatic hydrocarbons, e.g., benzene, toluene, etc.; chlorinated hydrocarbons, e.g., dichloromethane, chloroform, etc.; ethers, e.g., dioxane, diethyl ether, etc.; alcohols, e.g., methanoli amides such as dimethylformamide, etc.; dimethylsulfoxide; water;
or carboxylic acids such as formic acid, acetic acid, propionic acid, etc.
,~
. . ~ . - .
, 1 The reaction suitably proceeds at temperature above about -20C, bu-t in general, a temperature ranye between 0C and 70C is preferred for obtaining good results. Generally, the reaction is completed in a period of about 2 to 50 hours.
Applying the process of the present invention, the peptide of the formula (I) can be prepared in high yield by pr~tecting the amino group in an amino acid, peptide or derivative thereof temporarily with the phosphoryl group of the formula (III), con-verting them to the desired peptides which still have this pro-tective group, and then eliminating the amino protective group with a phosphorus acid under mild conditions.
Furthermoxe, as is weIl known in general, it is very important for ~solation and purification of a compound containing functional group to protect the functional group temporarily, and in this respect the present invention is also very useful for achieving such a purpose. For example, in order to obtain a compound of the formula (I) in high purity, this purification can be achieved by converting the impure compound of the formula (I) to the phosphoryl derivative of the formula (II) by a well-known method, purifying the phosphoryl derivative of the formula (II), and then de-phosphorylating the derivative of the formula (II! to the compound of the formula (I) by the process of this invention.
The peptide of the formula (II) can be prepared by a conventional process per se in the art of the present invention, ;~
for instance, they can be prepared by reacting an amino acid or ~`~
a peptide of the formula (IV)~
A2 ~ NH2 (IV) 1083~)9 1 which consti-tutes the peptide of the formula (I) together with an additional amino acid or peptide or which is the peptide of the formula (I), with a compound of the formul~ (v):
R10 ~ P ~ X . (V) ~ , wherein Rl and R2 are each as defined above; and X is a halogen atom, to produce a compound of the formula (VI):
O
R 0 - P - NH - A ~VI) OR2 ~: , wherein Rl and R2 are each as defined above; and A2 is the residual group of the amino acid or peptide o~ the formula (IV), and, if necessary, then expanding ~2 to Al by condensation of the compound oE the formula (VI) with an additional amino acid or peptide.
The condensation can be carried out employing various condensation methods which are used in the axt, for example, an acid halide method, a mixed acld anhydride method and a reactive ester method.
In the synthesis of the compounds represented by formulas (II) and (IV), it is of course necessary to previously protect functional groups which adversely affect the desired reaction with a suitable protective group as used in the art.
The following examples are given to illustrate the invention more specifically, but are not in any way to be construed as limiting the scope o~ the present invention.
3~ Unless otherwise indicated herein, all parts, percents, ratios and the like are by weight. ~-~ _ 9 _ ~;)81~09 1 FX~MPLE`~ l Preparation of 7-(D-a-Aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylic ~cid Step A:
In llO mQ of an aqueous lN-sodium hydroxide solution was dissolved 15 g of D-phenylglycine. To the resulting solution was added dropwise 20 g of diJnethylphosphoryl chloride at room temperature (i.e., 20~ 30C) while keeping the pH of the reaction mixture at 9 to ll with the addition o an aqueous 2N-sodium hydroxide solution.
The reaction mixture was stirred for an additional 20 minutes, washed with diethyl ether, adiusted to a pH of 2 with conc. hydrochloric acid and then extracted with dichloromethane.
The organic layer was washed with water, dried over magnesium sulfate and concentrated to obtain 16 g of oily D-a-(dimethyl-phosphoramido)phenylacetic acid.
Q ) IR: vmax (neat) 1725 cm 1 Three grams of the product thus-obtained was dissolved ~ .
in 30 mQ of dichloromethane and then 3 g of thionyl chloride was ~ ~;
added thereto. The resulting solution was heated under reflux for 1 hour. The reaction solution was concentrated under reduced pressure to obtain 3.2 g of oily D-~-(dimethylphosphoramido)phenyl~
acetyl chlorlde. The product thus-obtained showed a strong absorption due to a carbonyl group (-COCQ) at 1805 cm l in the IR spectrum.
To a suspension of 2 g of 7~amino-3-methyl-ceph-3-em-4-carboxylic acid in 30 mQ of water 2.4 g of sodium bicarbonate was added to dissolve the acid. The resulting solution was cooled to 0 to 5C and a solution of 3 g of the acid chloride 1 ~ -.
.... . . . . , . . . . ,,, .. , ,, ~ . .... .. .
10~1~(19 1 obtained above in 20 rnQ of ace-tone was added dropwise thereto while stirrincJ. The reaction mixture was stirred for an additional 20 minutes, adjusted ~o a pll of 1 with addition of conc. hydro-chloric acid and extracted with dichloromethane. The separated dichloromethane layer was dried over magnesium sulfate and concentrated. Diethyl ether was added to the residue and the precipitated crystals were filtered to obtain 2.3 g of 7-[D-a-(dimethylphosphoramido)phenylacetamido]-3-methyl-ceph-3-em-4-carboxylic acid (m.p. 190 ~ 192C).
1CI :
[~D~ +78 (c = 1, EtOH) IR: vmax (Nujol) 1780, 1715, 1660 cm Step B:
Two grams of 7-[D-~-(dimethylphosphoramido)phenyl-acetamido]-3-methyl-ceph-3-em-4-carboxylic acid which wa~ obtained in Step A above was dissolved in 5 mQ o~ 85% phosphoric acid, and the resulting mixture was stirred at room temperature for
5 hours.
After 100 mQ of diethyl ether was added to the reaction ~o solution, the mixture was stirred vigorously for S minutes and allowed to stand. The ether layer was removed by decantation and the lower layer was washed again with diethyl ether by the same operation. Then, the washed layer was dissolved in 5 mQ
of water and the solution was adjusted to a pH of 4.3 with an aqueous 2N-sodium hydroxide solution. Then 15 mQ of acetone was added thereto and the resulting mixture was allowed to stand overnight at O to 5C. The precipitated crystals were filtered, washed with a small amount of water and acetone and dried under -reduced pressure to obtain 1.2 g of 7-(D-~-aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylic acid as white crystals. The results of IR and TLC analysis of the product thus-obtained agreed completely those of an authentic sample.
,~,1 - 11 -~C~ ] D -1-151 (C = O . 5 r ~2) Preparation of 2,2,2-Trichloroethyl 7-(D-~-Aminophenyl-acetamido)-3-methyl-ceph-3-em-4-carboxylate . . . , ~
Step A:
In 10 mQ of dichloromethane were dissolved 1.36 g of D-~-(dimethylphosphoramido)phenylacetic acid and 0.41 g of N-methyl morpholine and the resultiny mixture was cooled to -15 to -10C. To the mixture was added 0.43 g of ethyl chlorocarbonate and, after stirring the mixture for 10 minutes at the same temperature, 1.53 g of 2,2,2-trichloroethyl 7-amino-3-methyl-ceph-3-em-4-carboxylate hydrochloride was added thereto. There-after, additional N-methyl morpholine (0.41 g) was added drop-wise over a 15 minute period. After stirring the reaction ~. .
solution for 1 hour at the same temperature, the solution was :, , washed successively with lN-hydrochloric acid and a saturated aqueous sodium bicarbonate solution, dried over magnesium sulfate and concentrated. The residue was recrystallized from a mixed 2~ solvent of isopropyl alcohol and n-hexane to obtain 1.89 g of ;
2,2,2-trichloroethyl 7-[D-a-(dimethylphoSphoramido)phenylacet-amido]-3-methyl-ceph-3-em-4-carboxylate (m.p. 99 ~ 102C).
IR: vmax (Nujol~ 1780, 1740, 1680 cm 1 ;~
Step B:
In S g of 85% phosphoric acid was dissolved 1.5 g of 2,2,2-trichloroethyl 7-~D-~-(dimethylphosphoramido)phenylacet- ;
amido]-3-methyl-ceph-3-em-4-carboxylate which was obtained in Step A above, and the resulting mixture was stirred for 10 hours at room temperature. After adding 30 mQ of water to the reaction mixture, the mixture was adjusted to a pH of 6 with a 2N a~ueous Trade ~lark l2~9 1 sodium hydroxide solution and extracted wi-th ethyl acetate.
The ethyl aceta-te layer was washed with water, dried over magnesium sulEa-te ancl concentrated to obtain 1.16 g of 2,2,2-trichloroethyl 7-(D-U-aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylate as a pale yellow solid.
IR: vmax (CEICQ3) 1780, 1740, 1685 cm 1 NMR tCDCQ3) ~: 1.96 ~2H, singlet, NH2), 2~21 (3EI, singlet, 3-CH3), 3.40 (2H, multiplet, -S-C~2-), 5.78 (lH, multiplet, 7-H), 7.36 (5H, singlet, aromatic ring), 7.96 (lH, doublet, J=lOHz, -CONH-).
Preparation of Phenacyl 7-(D-~-Aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxyla-te Hydrochloride _ _ . . . . ~ . _ .. _ . .. . _ . .. .. _ Step A:
To 110 mQ of a lN aqueous sodium hydroxi~e solution was added 15 g of D-phenylglycine. To the mlxture was added dropwise 20 g of diisopropylphosphoryl chloride at room temperature, while keeping the pH of the reaction mixture at 9 to 11 by the addition of a 2N aqueous sodium hydroxide solution.
The reaction mixture was stirred for an additional 20 minutes, cooled to O to 5C, and then adjusted to a pH of 2 with conc. hydrochloric acid. After the reaction solution was stirred for 30 minutes at the same temperature, the precipitated crystals were filtered and dried over phosphorus pentoxide under reduced pressure to obtain 30 g of D-a-(diisopropylphosphoramido)phenyl-acetic acid (m.p. 124 ~ 126C).
[~]20 -144 (c = 1 CHCQ ) 1(3 8~
1 In 30 mQ o~ dichlorome-thane were dissolved 3.15 y of the carboxylic acid thus-obtained above and 1.04 g o~ triethyl~
amine, ancl to the resulting solution 1.12 g of ethyl chloro-carbonate was added drcpwise at -15 to -10C while stirriny. The reaction mixture was stirred at the same temperature for an additional 10 minutes. Then, 3.69 g of phenacyl 7-amino-3-methyl-ceph-3-em-4-carboxylate hydrochloride was added thereto and sub-sequently 1.01 g of triethylamine was added dropwise thereto over a 15 minute period. After the mixture was stirred at the same 1~ temperature for an additional hour, the reaction mixture was washed successively with lN-hydrochloric acid and a saturated aqueous sodium bicar~onate solution, dried over magnesium sulfate and concentrated. The residue was recrystallized from a mixed solvent of dichloromethane and ethyl acetate to obtain 5.86 ~
of phenacyl 7[D-~-(diisopropylphosphoramido~phenylacetamido]-3-methyl-ceph--3-em-4-carboxylate (m.p. 224 ~ 225C).
IR: vmax (Nu~ol) 1785, 1740, 1685, 1660 cm 1 Step B:
~ In 10 mQ of polyphosphoric acid was dissolved 3 g of phenacyl 7-[D-a-(diisopropylphosphoramido)phenylacetamido~-3-methyl-ceph-3-em-4-carboxylate, and the resulting mixture was stirred at room temperature for 10 hours. After adding 50 mQ
of water to the reaction mixture, the mixture was adjusted to a pH of 6 by the addition of a 2N aqueous sodium hydroxide solution and then extracted with dichloromethane~ The dichloro-methane layer was cooled to 0 to 5C and then 5 mQ of a 3N-hydrochloric acid was added thereto while stirring. After the solution was stirred for 3 hours at the same temperature, the 3~ precipitated crystals were filtered to obtain 2.2 g of phenacyl .
' .... ~; .
~08~2~ ~
1 7-(D-C~-aminophenylacetamido)-3-methyl-c~ph-3-em-4-carbox~lait~
hydrochloride (m.p. 178 ~ 180C).
IR: vmax (Nujol) 1762, 1718, 1690, 1670 cm 1 Preparation of 7-(D-~-Aminophenylacetamido)-3-m~thyl-~ . , Step A:
In 30 mQ of for~ic acid was dissolved 3 g of phenacyl 7-[D-~-(diisopropylphosphoramido)phenylacetamido~-3-methyl-eeph-3-em-4-carboxylate. To the resulting mixture was added 1.5 g of zinc powder, and the mixture was stirred at 5 to 10C for 2 hours.
After completion of the reaction, the exeess zinc powder was filtered off and the filtrate was coneentrated under reduced pressure. To the resulting residue was added 30 mQ of lN-hydrochloric acid and the mixture was extracted with dichloro-methane. The organic layer was washed with water, dried over magnesium sulfate and concentrated. Diethyl ether was added to the residue, and the preipitated crystals were filtered to obtain 2.1 g of 7-[D-~-(diisopropylphosphoramido)phenylacetamido~-3-methyl-ceph-3-em~4-carboxylic acid (m.p. 146 ~ 147C).
E~25 ~53 (e = 1, EtOH) IR: vmax (Nujol) 1790, 1720, 1665 cm 1 Step B:
.
In 5 mQ of 85~ phosphoric acid was dissolved 2 g of 7-[D-a-~diisopropylphosphoramido)phenylacetamido}-3-methyl-ceph-3-em-4-carboxylie acid, and the resulting mixture was stirred at room temperature for 8 hours. The solution was treated in the same manner as described in Example 1 to obtain 1.0 g of 7-(D-~-- 15 - ;~
,, .
~L0~31%~ ~
-1 aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylic acid. The IR spectrum oE the product thus-obtained agreed completely with that of an authentic sample.
[~]D ~148 (c = 0.5, H20) Preparation of L-Alanyl-L-leucine Methyl Ester Hydrochloride In 10 g of polyphosphoric acid was dissolved 3 g of ~;
N-[bis-(p-nitrobenzyl)phosphoryl]-L-alanyl-L-leucine methyl ester (which was prepared as disclosed in Chem. Ber., 94, 2644 (1961)), and the reaction mixture was allowed to stand at room tempe~ature ~-for 18 hours.
After the reaction was completed, 30 mQ of water was added. The precipitated crystals (which were identi~ied with bis-(p-nitrobenzyl)phosphate on the basis of elemental analysis and the IR spectrum and the melting point were in complete agree~
ment those of an authentic sample) were filtered, and the filtrate was neutralized with solid sodium bicarbonate and extracted with ~ ~;
ethyl acetate. To the separated ethyl acetate layer was added 1 mQ of conc. hydrochloric acid, and the mixture was concentrated to half of the original volume. The precipitated crystals were filtered to obtain 1.2 g of L-alanyl-L-leucine methyl ester hydrochloride.
[~]20 ~5.5 (c = 1, methanol) m.p. 178 ~ 179C
.;~ ~':
Preparation of S-Benzyl-L-cysteinylglycine Step A: ; ' In 80 mQ of O.lN aqueous sodium hydroxide was dissolved
After 100 mQ of diethyl ether was added to the reaction ~o solution, the mixture was stirred vigorously for S minutes and allowed to stand. The ether layer was removed by decantation and the lower layer was washed again with diethyl ether by the same operation. Then, the washed layer was dissolved in 5 mQ
of water and the solution was adjusted to a pH of 4.3 with an aqueous 2N-sodium hydroxide solution. Then 15 mQ of acetone was added thereto and the resulting mixture was allowed to stand overnight at O to 5C. The precipitated crystals were filtered, washed with a small amount of water and acetone and dried under -reduced pressure to obtain 1.2 g of 7-(D-~-aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylic acid as white crystals. The results of IR and TLC analysis of the product thus-obtained agreed completely those of an authentic sample.
,~,1 - 11 -~C~ ] D -1-151 (C = O . 5 r ~2) Preparation of 2,2,2-Trichloroethyl 7-(D-~-Aminophenyl-acetamido)-3-methyl-ceph-3-em-4-carboxylate . . . , ~
Step A:
In 10 mQ of dichloromethane were dissolved 1.36 g of D-~-(dimethylphosphoramido)phenylacetic acid and 0.41 g of N-methyl morpholine and the resultiny mixture was cooled to -15 to -10C. To the mixture was added 0.43 g of ethyl chlorocarbonate and, after stirring the mixture for 10 minutes at the same temperature, 1.53 g of 2,2,2-trichloroethyl 7-amino-3-methyl-ceph-3-em-4-carboxylate hydrochloride was added thereto. There-after, additional N-methyl morpholine (0.41 g) was added drop-wise over a 15 minute period. After stirring the reaction ~. .
solution for 1 hour at the same temperature, the solution was :, , washed successively with lN-hydrochloric acid and a saturated aqueous sodium bicarbonate solution, dried over magnesium sulfate and concentrated. The residue was recrystallized from a mixed 2~ solvent of isopropyl alcohol and n-hexane to obtain 1.89 g of ;
2,2,2-trichloroethyl 7-[D-a-(dimethylphoSphoramido)phenylacet-amido]-3-methyl-ceph-3-em-4-carboxylate (m.p. 99 ~ 102C).
IR: vmax (Nujol~ 1780, 1740, 1680 cm 1 ;~
Step B:
In S g of 85% phosphoric acid was dissolved 1.5 g of 2,2,2-trichloroethyl 7-~D-~-(dimethylphosphoramido)phenylacet- ;
amido]-3-methyl-ceph-3-em-4-carboxylate which was obtained in Step A above, and the resulting mixture was stirred for 10 hours at room temperature. After adding 30 mQ of water to the reaction mixture, the mixture was adjusted to a pH of 6 with a 2N a~ueous Trade ~lark l2~9 1 sodium hydroxide solution and extracted wi-th ethyl acetate.
The ethyl aceta-te layer was washed with water, dried over magnesium sulEa-te ancl concentrated to obtain 1.16 g of 2,2,2-trichloroethyl 7-(D-U-aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylate as a pale yellow solid.
IR: vmax (CEICQ3) 1780, 1740, 1685 cm 1 NMR tCDCQ3) ~: 1.96 ~2H, singlet, NH2), 2~21 (3EI, singlet, 3-CH3), 3.40 (2H, multiplet, -S-C~2-), 5.78 (lH, multiplet, 7-H), 7.36 (5H, singlet, aromatic ring), 7.96 (lH, doublet, J=lOHz, -CONH-).
Preparation of Phenacyl 7-(D-~-Aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxyla-te Hydrochloride _ _ . . . . ~ . _ .. _ . .. . _ . .. .. _ Step A:
To 110 mQ of a lN aqueous sodium hydroxi~e solution was added 15 g of D-phenylglycine. To the mlxture was added dropwise 20 g of diisopropylphosphoryl chloride at room temperature, while keeping the pH of the reaction mixture at 9 to 11 by the addition of a 2N aqueous sodium hydroxide solution.
The reaction mixture was stirred for an additional 20 minutes, cooled to O to 5C, and then adjusted to a pH of 2 with conc. hydrochloric acid. After the reaction solution was stirred for 30 minutes at the same temperature, the precipitated crystals were filtered and dried over phosphorus pentoxide under reduced pressure to obtain 30 g of D-a-(diisopropylphosphoramido)phenyl-acetic acid (m.p. 124 ~ 126C).
[~]20 -144 (c = 1 CHCQ ) 1(3 8~
1 In 30 mQ o~ dichlorome-thane were dissolved 3.15 y of the carboxylic acid thus-obtained above and 1.04 g o~ triethyl~
amine, ancl to the resulting solution 1.12 g of ethyl chloro-carbonate was added drcpwise at -15 to -10C while stirriny. The reaction mixture was stirred at the same temperature for an additional 10 minutes. Then, 3.69 g of phenacyl 7-amino-3-methyl-ceph-3-em-4-carboxylate hydrochloride was added thereto and sub-sequently 1.01 g of triethylamine was added dropwise thereto over a 15 minute period. After the mixture was stirred at the same 1~ temperature for an additional hour, the reaction mixture was washed successively with lN-hydrochloric acid and a saturated aqueous sodium bicar~onate solution, dried over magnesium sulfate and concentrated. The residue was recrystallized from a mixed solvent of dichloromethane and ethyl acetate to obtain 5.86 ~
of phenacyl 7[D-~-(diisopropylphosphoramido~phenylacetamido]-3-methyl-ceph--3-em-4-carboxylate (m.p. 224 ~ 225C).
IR: vmax (Nu~ol) 1785, 1740, 1685, 1660 cm 1 Step B:
~ In 10 mQ of polyphosphoric acid was dissolved 3 g of phenacyl 7-[D-a-(diisopropylphosphoramido)phenylacetamido~-3-methyl-ceph-3-em-4-carboxylate, and the resulting mixture was stirred at room temperature for 10 hours. After adding 50 mQ
of water to the reaction mixture, the mixture was adjusted to a pH of 6 by the addition of a 2N aqueous sodium hydroxide solution and then extracted with dichloromethane~ The dichloro-methane layer was cooled to 0 to 5C and then 5 mQ of a 3N-hydrochloric acid was added thereto while stirring. After the solution was stirred for 3 hours at the same temperature, the 3~ precipitated crystals were filtered to obtain 2.2 g of phenacyl .
' .... ~; .
~08~2~ ~
1 7-(D-C~-aminophenylacetamido)-3-methyl-c~ph-3-em-4-carbox~lait~
hydrochloride (m.p. 178 ~ 180C).
IR: vmax (Nujol) 1762, 1718, 1690, 1670 cm 1 Preparation of 7-(D-~-Aminophenylacetamido)-3-m~thyl-~ . , Step A:
In 30 mQ of for~ic acid was dissolved 3 g of phenacyl 7-[D-~-(diisopropylphosphoramido)phenylacetamido~-3-methyl-eeph-3-em-4-carboxylate. To the resulting mixture was added 1.5 g of zinc powder, and the mixture was stirred at 5 to 10C for 2 hours.
After completion of the reaction, the exeess zinc powder was filtered off and the filtrate was coneentrated under reduced pressure. To the resulting residue was added 30 mQ of lN-hydrochloric acid and the mixture was extracted with dichloro-methane. The organic layer was washed with water, dried over magnesium sulfate and concentrated. Diethyl ether was added to the residue, and the preipitated crystals were filtered to obtain 2.1 g of 7-[D-~-(diisopropylphosphoramido)phenylacetamido~-3-methyl-ceph-3-em~4-carboxylic acid (m.p. 146 ~ 147C).
E~25 ~53 (e = 1, EtOH) IR: vmax (Nujol) 1790, 1720, 1665 cm 1 Step B:
.
In 5 mQ of 85~ phosphoric acid was dissolved 2 g of 7-[D-a-~diisopropylphosphoramido)phenylacetamido}-3-methyl-ceph-3-em-4-carboxylie acid, and the resulting mixture was stirred at room temperature for 8 hours. The solution was treated in the same manner as described in Example 1 to obtain 1.0 g of 7-(D-~-- 15 - ;~
,, .
~L0~31%~ ~
-1 aminophenylacetamido)-3-methyl-ceph-3-em-4-carboxylic acid. The IR spectrum oE the product thus-obtained agreed completely with that of an authentic sample.
[~]D ~148 (c = 0.5, H20) Preparation of L-Alanyl-L-leucine Methyl Ester Hydrochloride In 10 g of polyphosphoric acid was dissolved 3 g of ~;
N-[bis-(p-nitrobenzyl)phosphoryl]-L-alanyl-L-leucine methyl ester (which was prepared as disclosed in Chem. Ber., 94, 2644 (1961)), and the reaction mixture was allowed to stand at room tempe~ature ~-for 18 hours.
After the reaction was completed, 30 mQ of water was added. The precipitated crystals (which were identi~ied with bis-(p-nitrobenzyl)phosphate on the basis of elemental analysis and the IR spectrum and the melting point were in complete agree~
ment those of an authentic sample) were filtered, and the filtrate was neutralized with solid sodium bicarbonate and extracted with ~ ~;
ethyl acetate. To the separated ethyl acetate layer was added 1 mQ of conc. hydrochloric acid, and the mixture was concentrated to half of the original volume. The precipitated crystals were filtered to obtain 1.2 g of L-alanyl-L-leucine methyl ester hydrochloride.
[~]20 ~5.5 (c = 1, methanol) m.p. 178 ~ 179C
.;~ ~':
Preparation of S-Benzyl-L-cysteinylglycine Step A: ; ' In 80 mQ of O.lN aqueous sodium hydroxide was dissolved
6.3 g of S-benzyl-L-cysteine, and 5.7 g of dimethylchlorophosphate ~ - 16 -i - . ~ :' - - ~ .. . . ... . . . .
12()9 1 was added dropwise a-t 25 to 30C over a 30 minu-te period followed by stirring at the same temperature for 30 minutes. The reaction mixture was washed with diethyl ether, acidified with a 6N aqueous hydrochloric acid solution to a pH of 2 and extracted with di-chloromethane. The dichloromethane layer was washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 6.7 g of S-benzyl-N-dimeth~lphosphoryl-L-cysteine as a colorless oil.
IR: vmax (film) 1735 cm 1 [~l25 ~28 (c = 1, CEICQ3) To a mixture o~ 50 mQ of dichloromethane, 6.4 g of the product thus-obtained, 2.8 g of glycine ethyl ester hydrochloride and 4.1 g of triethylamine was added 4.5 g of dicyclohexylcarbo-diimide. The mixture was allowed to stand at room temperature for 20 hours. After the precipitate was removed by filtration, the reaction mixture was washed successively with a lN aqueous hydrochloric acid solution and a 5% aqueous potassium bicarbonate solution, dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 7.3 g of (S-benzyl-N-dimethyl-phosphoryl)-L-cysteinylglycine ethyl ester as an oil.
IR: vmax (film) 1740, 1655 cm 1 The product thus-obtained was used for the next step without further purification.
Step B:
In 30 g of 85% phosphoric acid was dissolved 6.1 g of the product obtained in Step A above, and the mixture was stirred at room temperature for 20 hour~. After 100 mQ of water was added, the reac-tion mixture was adjusted to a pH o~ 7 with a 30%
aqueous sodium hydroxide solution and extracted with ethyl acetate.
The separated e~hyl acetate layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 4.5 g of S-benzyl-L-cys-teinylglycine ethyl es-ter as an oil.
IR: vmax (film) 1740, 1650 cm Saponification of the product thus-obtained by treatment with a methanolic sodium hydroxide solution produced 3.8 g of S-benzyl-L-cysteinylglycine.
m.p. 163 ~ 164C
~D ~28 (c = 1, in lN NaOH) While the invention has been described in detail and with reEerence to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modiPications can be made therein without departing from the spirit and scope thereof.
3~ .
- 13 - ~:
12()9 1 was added dropwise a-t 25 to 30C over a 30 minu-te period followed by stirring at the same temperature for 30 minutes. The reaction mixture was washed with diethyl ether, acidified with a 6N aqueous hydrochloric acid solution to a pH of 2 and extracted with di-chloromethane. The dichloromethane layer was washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 6.7 g of S-benzyl-N-dimeth~lphosphoryl-L-cysteine as a colorless oil.
IR: vmax (film) 1735 cm 1 [~l25 ~28 (c = 1, CEICQ3) To a mixture o~ 50 mQ of dichloromethane, 6.4 g of the product thus-obtained, 2.8 g of glycine ethyl ester hydrochloride and 4.1 g of triethylamine was added 4.5 g of dicyclohexylcarbo-diimide. The mixture was allowed to stand at room temperature for 20 hours. After the precipitate was removed by filtration, the reaction mixture was washed successively with a lN aqueous hydrochloric acid solution and a 5% aqueous potassium bicarbonate solution, dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 7.3 g of (S-benzyl-N-dimethyl-phosphoryl)-L-cysteinylglycine ethyl ester as an oil.
IR: vmax (film) 1740, 1655 cm 1 The product thus-obtained was used for the next step without further purification.
Step B:
In 30 g of 85% phosphoric acid was dissolved 6.1 g of the product obtained in Step A above, and the mixture was stirred at room temperature for 20 hour~. After 100 mQ of water was added, the reac-tion mixture was adjusted to a pH o~ 7 with a 30%
aqueous sodium hydroxide solution and extracted with ethyl acetate.
The separated e~hyl acetate layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 4.5 g of S-benzyl-L-cys-teinylglycine ethyl es-ter as an oil.
IR: vmax (film) 1740, 1650 cm Saponification of the product thus-obtained by treatment with a methanolic sodium hydroxide solution produced 3.8 g of S-benzyl-L-cysteinylglycine.
m.p. 163 ~ 164C
~D ~28 (c = 1, in lN NaOH) While the invention has been described in detail and with reEerence to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modiPications can be made therein without departing from the spirit and scope thereof.
3~ .
- 13 - ~:
Claims (6)
1. A process for producing a peptide derivative of the formula (I):
A1 - NH2 (I) wherein A1 is a residual part of a peptide derivative made from at least two amino acid compounds selected from the group consis-ing of glycine, alanine, valine, norvaline, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, sarcosine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, ornithine, arginine, phenylalanine, tyrosine, histidine, triptophan, proline, hydroxyproline, .alpha.-aminobutyric acid, .gamma.-aminobutyric acid .alpha.-.gamma.-diaminobutyric acid, .alpha.-aminoadipic acid, .alpha.-phenylglycine, .alpha.-p-hydroxyphenylglycine, .alpha.-p-chloro-phenylglycine, .alpha.-(2-thienyl)-glycine, .alpha.-(2-furyl)glycine, .alpha.-(1-cyclohexenyl)glycine, (1-cyclohexadienyl)glycine, 7-amino-4-carboxy-3-methyl-3-cephem, and their protected derivatives, which comprises reacting a phosphoramide derivative of the formula (II):
(II) wherein R1 and R2 are each C1 - C6 alkyl, C3 - C7 cycloalkyl, phenyl p-tolyl, p-chlorophenyl, benzyl, p-nitrobenzyl or p-chlorobenzyl, and A1 is as defined hereinbefore, with ortho-phosphoric acid, phosphorous acid or polyphosphoric acid.
A1 - NH2 (I) wherein A1 is a residual part of a peptide derivative made from at least two amino acid compounds selected from the group consis-ing of glycine, alanine, valine, norvaline, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, sarcosine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, ornithine, arginine, phenylalanine, tyrosine, histidine, triptophan, proline, hydroxyproline, .alpha.-aminobutyric acid, .gamma.-aminobutyric acid .alpha.-.gamma.-diaminobutyric acid, .alpha.-aminoadipic acid, .alpha.-phenylglycine, .alpha.-p-hydroxyphenylglycine, .alpha.-p-chloro-phenylglycine, .alpha.-(2-thienyl)-glycine, .alpha.-(2-furyl)glycine, .alpha.-(1-cyclohexenyl)glycine, (1-cyclohexadienyl)glycine, 7-amino-4-carboxy-3-methyl-3-cephem, and their protected derivatives, which comprises reacting a phosphoramide derivative of the formula (II):
(II) wherein R1 and R2 are each C1 - C6 alkyl, C3 - C7 cycloalkyl, phenyl p-tolyl, p-chlorophenyl, benzyl, p-nitrobenzyl or p-chlorobenzyl, and A1 is as defined hereinbefore, with ortho-phosphoric acid, phosphorous acid or polyphosphoric acid.
2. A process as claimed in claim 1 wherein said reacting is at a temperature of from about 0°C to about 70°C.
3. A process as claimed in claim 1 wherein the reacting is in a mixture of said derivative of the formula (II) and ortho-phosphoric acid, phosphorous acid or polyphosphoric acid or in a solution or suspension in an inert solvent of said derivative of the formula (II) and ortho-phosphoric acid, phosphorous acid or polyphosphoric acid.
4. A process as claimed in claim 3 wherein said inert solvent is selected from the group consisting of an aromatic hydrocarbon, an aliphatic chlorinated hydrocarbon, an ether, an alcohol, an amide, water and a carboxylic acid.
5. A process as claimed in claim 4 wherein said inert solvent is selected from the group consisting of benzene, toluene, dichloromethane, chloroform, dioxane, diethyl ether, methanol, dimethylformamide, water and acetic acid.
6. A process as claimed in claim 1 wherein the amount of said phosphorous acid is greater than 1 mole of ortho-phosphoric acid, phosphorous acid or polyphosphoric acid per mole of the derivative of the formula (II).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP49108985A JPS5136489A (en) | 1974-09-20 | 1974-09-20 | |
| JP6123375A JPS51136602A (en) | 1975-05-21 | 1975-05-21 | Process for preparation of amino acid or peptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1081209A true CA1081209A (en) | 1980-07-08 |
Family
ID=26402286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA235,873A Expired CA1081209A (en) | 1974-09-20 | 1975-09-19 | Process for the preparation of peptides |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US4043991A (en) |
| CA (1) | CA1081209A (en) |
| CH (1) | CH606009A5 (en) |
| DE (1) | DE2541953A1 (en) |
| FR (2) | FR2292699A1 (en) |
| GB (1) | GB1519997A (en) |
| NL (1) | NL7511074A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5941999B2 (en) * | 1975-07-24 | 1984-10-11 | 武田薬品工業株式会社 | Method for producing cefem or venam compounds |
| ES479377A1 (en) * | 1978-04-07 | 1979-12-01 | Glaxo Group Ltd | Process for preparing 3-carbamoyloxymethyl cephalosporins |
| PT69450A (en) * | 1978-04-07 | 1979-05-01 | Glaxo Group Ltd | Improvements in or relating to cephalosporin compounds |
| DE2901843A1 (en) * | 1979-01-18 | 1980-07-31 | Hoechst Ag | METHOD FOR PRODUCING CARBONIC ACID AMIDES AND PEPTIDES |
| US4291031A (en) * | 1979-02-19 | 1981-09-22 | Fujisawa Pharmaceutical Co., Ltd. | 3-Phosphonocephalosporanic acid derivatives, and pharmaceutical composition comprising the same |
| JPS55136289A (en) * | 1979-04-06 | 1980-10-23 | Glaxo Group Ltd | Manufacture of cephalosporin compound |
| FR2511372A1 (en) * | 1981-08-17 | 1983-02-18 | Rhone Poulenc Sante | NEW DERIVATIVES OF CEPHALOSPORINE AND THEIR PREPARATION |
| GB2106114B (en) * | 1981-09-25 | 1985-06-12 | Wellcome Found | Phosphorus - containing amides |
| US5390915A (en) * | 1992-01-23 | 1995-02-21 | Levatino; Samuel R. | Baseball batting training machine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4970989A (en) * | 1972-11-15 | 1974-07-09 | ||
| US3980644A (en) * | 1973-04-23 | 1976-09-14 | Eli Lilly And Company | Pentavalent phosphorus amides of cephalosporins and separation process using same |
-
1975
- 1975-09-18 GB GB38457/75A patent/GB1519997A/en not_active Expired
- 1975-09-19 CA CA235,873A patent/CA1081209A/en not_active Expired
- 1975-09-19 DE DE19752541953 patent/DE2541953A1/en not_active Withdrawn
- 1975-09-19 CH CH1221375A patent/CH606009A5/xx not_active IP Right Cessation
- 1975-09-19 NL NL7511074A patent/NL7511074A/en not_active Application Discontinuation
- 1975-09-22 US US05/615,751 patent/US4043991A/en not_active Expired - Lifetime
- 1975-09-22 FR FR7529000A patent/FR2292699A1/en active Granted
-
1976
- 1976-05-04 FR FR7613273A patent/FR2303020A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| FR2292699A1 (en) | 1976-06-25 |
| FR2303020A1 (en) | 1976-10-01 |
| FR2303020B1 (en) | 1979-04-06 |
| CH606009A5 (en) | 1978-10-13 |
| NL7511074A (en) | 1976-03-23 |
| GB1519997A (en) | 1978-08-02 |
| FR2292699B1 (en) | 1979-02-02 |
| US4043991A (en) | 1977-08-23 |
| DE2541953A1 (en) | 1976-04-01 |
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